This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.


Sections of Whole Mount in situ Hybridization Preparations

(Source: R. BreMiller)

Resin Sections

1. Use embryos in which alkaline phosphatase has been employed as a detection enzyme. For the best results it is important to overstain heavily the whole-mount preparation.

2. Postfix embryos in 4% paraformaldehyde in PBS. The tissue can be stored in this solution for an extended period of time in the dark at 4°C.

3. Wash embryos in dH2O and dehydrate quickly through a graded series of methanol (50%, 70%, 95% and twice 100% 2 to 3 min each).

4. Replace the methanol with propylene oxide, two changes for 5 min each.

5. Replace with a 1:1 mixture of Epon and propylene oxide for 30 min.

6. Replace with a 3:1 mixture of Epon:propylene oxide for 2 to 3 hours.

7. Transfer tissue to pure Epon for 4 hours.

8. Embed in fresh Epon. Polymerize overnight in a 60°C oven.

9. Cut sections 5 to 10 µm thick with a glass knife. Dry down on a drop of water. Cover slip in Permount. Alternatively, cover slip in Epon and polymerize at 60°C.

Frozen sections

1. Wash stained embryos in PBS, two changes, 2 to 3 min each.

2. Wash in dH2O.

3. Embed embryos in agar-sucrose as described in Agar Mounting, Chapter 4. Cryoprotect in 30% sucrose and cut 14 to 16 µm sections in a -20°C cryostat. Pick up sections on subbed slides and dry at room temperature 2 to 3 hours.

4. Wash sections in dH2O 2 to 3 min and dehydrate through graded alcohols. Clear in xylene and mount in Permount.

NOTES: Aqueous solutions of 0.2% methyl green or 1% neutral red can be used as a counterstain for the frozen sections. Immerse slides briefly (30 to 90 sec) in the staining solution, wash in dH2O, and dehydrate as above.
The Zebrafish Book